We have developed a scheme for quantitative real-time (RTi) nucleic acid sequence-based amplification (NASBA) for the detection of Aspergillus fumigatus using internal control (IC) RNA. Construction of IC RNA began with the synthesis of nontarget sequences from Clavibacter michiganensis subsp. sepedonicus by a primary polymerase chain reaction (PCR) step, followed by a secondary PCR step using chimeric primers to produce a chimeric sequence including a T7 promoter region. Finally, chimeric IC RNAs were constructed by the use of in vitro transcription. The assay detected A. fumigatus within a range of 10(4) to 10(8) copies/mL of RNA and 10(0) to 10(8) cells. When the assay was performed with the target and IC RNA in 1 reaction in a single tube, there was little interference of the IC RNA in the measurement of the amount of target. The amount of RNA calculated using the assay was not significantly different from the amount of input RNA as indicated by Bland-Altman analysis. The IC RNA we constructed can be used in RTi-NASBA for quantitative detection of Aspergillus with good precision and accuracy.